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Characterization of Dickeya zeae isolates causing stalk rot of maize based on

biochemical assays and antibiotic sensitivity

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Indian Phytopath. 68 (4) : 375-379 (2015)

RESEARCH ARTICLE

Characterization of Dickeya zeae isolates causing stalk rot of

maize based on biochemical assays and antibiotic sensitivity

ADESH KUMAR, MANDEEP SINGH HUNJAN*, HARLEEN KAUR1 and P.P. SINGH
Department of Plant Pathology, 1Department of Plant Breeding and Genetics
Punjab Agricultural University, Ludhiana 141 004, Punjab, India

ABSTRACT: Maize growing areas of Punjab were surveyed during Kharif 2012 and 2013 for the occurrence of stalk rot
disease. A total of 59 isolates were isolated as off-white colonies on King’s B medium. The isolates showed variation in
virulence when tested on susceptible cv. Punjab Sweet Corn-1 and were grouped as highly virulent, moderately virulent and
less virulent. All the isolates produced cavities on crystal violet pectate medium. A panel of 27 biochemical tests were used
to characterize these isolates which showed differential reaction for utilization of starch and other carbohydrates, gelatin
liquefication and growth at high salt concentration. Multiple antibiotic resistance was observed in all the isolates tested.
Numerical analysis of phenotypic features revealed two major groups of isolates associated at 60 per cent similarity
coefficient.

Key words: Dickeya zeae, diversity, maize, stalk rot

Stalk rot caused by Dickeya zeae (formerly Erwinia
chrysanthemi pv. zeae), is an important bacterial disease
of maize that constraints its production significantly
(Thind and Payak, 1978). The pathogen is soil borne and
the disease is seen in Kharif sown crop under Punjab
conditions. The Kharif sown crop has the most
susceptible stage coinciding with the annual monsoon
rainfall in Punjab which aggravates the disease
development. The pathogen spreads from plant to plant
and field to field through rainwater and its runoff. Although
three bacterial pathogens have been reported to be
causing stalk rot of maize namely Erwinia dissolvens, E.
chrysanthemi pv. zeae and Pseudomonas syringae pv.
lapsa (Prasad, 1930; Hingorani et al., 1959; Sinha, 1966),
yet our previous studies have concluded that E.
chrysanthemi pv. zeae is predominantly associated with
this disease under Punjab conditions. Originally classified
under pectolytic Erwinia sp., the pathogen has been
recently re-classified as Dickeya zeae by Samson et al.
(2005), who proposed a major taxonomic change which
separated E. chrysanthemi into five species under the
new genus Dickeya. Initial symptoms of bacterial stalk
rot of maize include discoloration of the leaf sheath, which
spread rapidly up the stalk into the leaves. The external
symptom of bacterial stalk rot (BSR) is the maceration
of the stalk and basal internodes, resulting in the
softening and discoloration of infected tissues leading
to foul odor and ultimately the plant topples down
resulting in severe grain yield losses.
This bacterium has a wide host range causing soft
rots on other crops as well (Bradbury, 1986) which makes
it difficult to manage this bacterium (Goto, 1979). D. zeae
has been reported to be highly variable with respect to
biochemical, physiological, pathological and molecular
characters. No attempt so far has been made to classify
*Corresponding author: mandeep.hunjan@pau.edu
the different strains into races or pathotypes due to lack
of standard differentials in maize. In addition to long term
strategies to stalk rot disease management, it is also
important to assess the variation in D. zeae populations.
So far, there has been no comprehensive study on the
phenotypic variation in the population of D. zeae infecting
maize crop in Northern India. Phenotypic diversity refers
to relative differences in biochemical or physiological
attributes among different isolates of D. zeae besides
their virulence spectrum. Therefore, the present study
was undertaken to characterize the phenotypic diversity
prevalent in the Punjab populations of D. zeae infecting
maize.
MATERIALS AND METHODS
Collection of diseased samples
Different maize growing areas of Punjab viz. Hoshiarpur,
Shaheed Bhagat Singh Nagar, Jalandhar, Ropar,
Gurdaspur and Ludhiana were surveyed in the months
of July-August during kharif 2012 and 2013. At each
location, disease incidence and severity was recorded,
the infected stalks were put in plastic bags and kept on
ice. The samples were stored 4°C before further
processing.
Isolation of the pathogen
The isolation and the presence of the pathogen in the
infected sheath was ascertained microscopically for the
ooze from the cut ends, on a slide in a drop of sterile
water. The leaf sheath bits were surface sterilized by
immersing in 75% ethanol solution for one minute
followed by three washings with sterilized distilled water.
After surface sterilization, the leaf bits were cut and put
in sterile water on a slide and the bacterial ooze was
376 Indian Phytopathology 68 (4) : 375-379 (2015)

streaked on King’s B medium (HiMedia™, King et al., Gurdaspur Chak Kalan 2012 M40
1954). Single isolated colonies of the bacterium on the
Gurdaspur Jandwal 2012 M41
King’s B media were further purified, preserved in silica
Gurdaspur Purana Bhangala 2012 M42
gel and multiplied on the King’s B medium, as and when
required. Sixty two isolates of the bacterial stalk rot Gurdaspur Dhula Khera 2012 M43
pathogen, Dickeya zeae isolated from infected maize Gurdaspur Garna Sahib 2012 M44
stalks from different districts were designated as given Gurdaspur Tanda 2013 M45
in table 1. Gurdaspur Dhadhila 2013 M46
Gurdaspur Cholang 2013 M47
Pathogenicity test
Jalandhar Phillarpur 2012 M48
All the 59 isolates were grown individually in liquid King’s Ropar Behrampur 2012 M49
B media for 24 hrs and bacterial suspension was adjusted Ropar Shekhupur 2012 M50
Table 1. Designation of isolates of Dickeya zeae isolated from Ropar Tiprian 2012 M51
different maize growing areas of Punjab Ropar Chohal 2012 M52
District Location/Village Year Designation Ropar Haripur 2012 M53
Ropar Abiana 2012 M54
Ludhiana PAU 2013 M1
Ropar Nurpur Bedi 2012 M55
Ludhiana PAU 2013 M2
Ropar Ramgarh 2012 M56
Ludhiana PAU 2013 M3
Ropar Chak Guru 2012 M57
Ludhiana PAU 2013 M4
Ropar Chak Phullu 2012 M58
Ludhiana Rasulpur 2012 M5
Ropar Bora 2012 M59
Ludhiana Manuke 2012 M6
Ropar Bora 2012 M60
Ludhiana Bhamipura 2012 M7
Ludhiana Hathur 2012 M8
to approximately 108-109 cfu/ml. One month old plants of
Ludhiana Jagraon 2012 M9
maize cv. Punjab Sweet Corn-1 were inoculated with
Ludhiana Chakar 2012 M10 individual isolates of D. zeae using hypodermic syringe
Hoshiarpur Purheerah 2012 M11 in the 2nd internode from base of maize stalk (Rangarajan
Hoshiarpur Chabbewal 2012 M13 and Chakravarti, 1967). After inoculation, the plants were
Hoshiarpur Makhomazra 2012 M14 frequently irrigated to maintain high humidity and soil
Hoshiarpur Makhomazra 2012 M15
moisture which is important for disease development.
Initial disease symptoms were observed after four days
Hoshiarpur Bhopar 2012 M16
of inoculation and final observations were recorded after
Hoshiarpur Bhana 2012 M17 15 days of inoculation. Disease severity was recorded
Hoshiarpur Bahowal 2012 M18 by splitting the infected stalks longitudinally and scoring
Hoshiarpur Baghowal 2012 M19 the infected plant on 1-5 scale (Lal, 1981). The pathogen
Hoshiarpur Nangal 2012 M20 was re-isolated from the infected inoculated stalks and
SBS Nagar Bhan Mazra 2012 M21
reconfirmed as D. zeae.
SBS Nagar Makhupur 2012 M22
Biochemical characterization
SBS Nagar Mehadpur 2012 M23
SBS Nagar Ranewal Tapriyan 2012 M24 All the isolates were grown on nutrient broth. Inoculum
SBS Nagar Sahadara 2012 M25 of each strain was prepared by suspending bacterial
SBS Nagar Chankoan 2012 M26
growth in 10 ml of sterile distilled water and standardized
the concentration to108-109 cfu/ml. The biochemical tests
SBS Nagar Rurki Kurdh 2012 M27
were performed with standard procedures viz. starch
SBS Nagar Nagla 2012 M28 hydrolysis and gelatin liquification test (Cowan, 1974),
SBS Nagar Dhahan 2012 M29 acid formation from fructose, ribose, maltose and
SBS Nagar Karonwar 2012 M30 galactose (Schaad, 1980), anaerobic growth test (Hugh
SBS Nagar Jainpur 2012 M31 and Leifson, 1953) and tetrazolium salt tolerance test
SBS Nagar Hyatpur 2012 M32
(Lelliott and Stead, 1987). Twelve tests namely Indole,
methyl red, Voges Proskauer’s, citrate utilization, glucose,
Hoshiarpur Sarholan 2012 M33
adonitol, arabinose, lactose, sorbitol, mannitol, rhamnose
Hoshiarpur Mayiopatti 2012 M34 and sucrose utilization tests were done by using HiMVic®
Hoshiarpur Kukowal 2012 M35 biochemical kit. Susceptibility towards chloramphenicol
Hoshiarpur Kukowal 2012 M36 (30 mcg), tetracycline (30 mcg), ampicillin (10 mcg),
Hoshiarpur Narur Pashtaan 2012 M37 streptomycin (300 mcg) and penicillin-G (10 units)
Hoshiarpur Bhopar 2012 M38
antibiotics was also tested using HiMedia® antibiotics
discs. The phenotypic similarity between the various
Hoshiarpur Bhana 2012 M39
isolates was generated using unweighted paired group
Indian Phytopathology 68 (4) : 375-379 (2015)
Table 2. Disease severity score of different isolates on Punjab Sweet Corn-1 cultivar
Name of Isolates
M-7, M-9, M-13, M-14, M-29, Chilli-2 and Rice-1
M-1, M-2, M-3, M-4, M-5, M-6, M-8, M-10, M-11, M-16, M-19, M-20,
M-24, M-26, M-28, M-35, M-36, M-37, M-38, M-39, M-44, M-45, M-47,
M-50, M-52, M-56, M-57, M-58
M-15, M-17, M-18, M-21, M-22, M-23, M-25, M-27, M-30, M-31, M-32, M-33,
M-34, M-40, M-41, M-42, M-43, M-46, M-48, M-49, M-51, M-53, M-54, M-55,
M-59, M-60 and Chilli-1
mean averages using software programme PAST ver
2.1.5.
RESULTS AND DISCUSSION
Isolation and identification of pathogen
A total of 59 isolates of the bacteria were isolated from
diseased stalks (Table 1). Individual isolate was identified
on crystal violet pectate (CVP) medium on the basis of
cavity formation. These cultures were further purified on
King’s B Medium and the bacterium was fur ther
confirmed as off-white, slimy and shiny colonies
appeared on the medium.
Pathogenicity test
Significant variation in the aggressiveness in different
isolates of the pathogen was observed. Water soaked
symptoms started appearing after 24 hrs of inoculation
followed by rotting of the inoculated internodes in all the
isolates. Temporal observations on disease development
showed that four isolates namely M-7, M-9, M-13, M-14
and M-29, were found to be highly virulent and toppled
down the inoculated plants of cv. Punjab sweet Corn-1
within 4 to 5 days of inoculation. Out of sixty two, 28
isolates showed disease score ranging from 3-4 and
classified as moderately virulent (Table 2). These isolates
were unable to topple down the plant, however vertical
disease progress was observed upto the third internode
from the point of inoculation. The lowest disease score
of two was recorded in 27 isolates where the disease
progressed to almost half the inoculated internodes
(Table 2). The plants neither toppled down nor wilted and
the isolates were classified as less virulent. Other authors
have also reported that highly virulent isolates can topple
down plant within week of inoculation. Rangarajan and
Chakravarti (1967) demonstrated that inoculated plants
started showing symptoms at 2-4 days after inoculation
and at the end of 6th day, the plants collapsed at the
point of inoculation accompanied by rotting. Similarly,
Ferreira-Pinto et al. (1994) suggested syringe method
of inoculation and observed rotting type of symptoms
within 12 days of inoculation.
Frequency of positive biochemical reactions
Significant variations in the substrate utilization pattern
were observed in different isolates of D. zeae. It was
377
No. of Disease score Remarks
isolates Mean Range
4.0 5.0 5.0 Highly Virulent
28 3.28 3.0-4.0 Moderately
Virulent
27 2.0 2.0 Less Virulent
observed (Fig. 1) that majority of the isolates could utilize
most of the carbohydrates tested while differential pattern
with respect to six biochemical tests and one antibiotic
was otherwise noticed. It was observed that only 43.5%
isolates could grow at high salt concentration (5% NaCl)
and only 2 isolates could hydrolyze starch in the media.
Majority of the isolates were resistant to all the antibiotics
tested while 43.5% isolates showed resistance to
streptomycin at 300mcg concentration. Similarly, it was
also found that half of the isolates could utilize gelatin in
the media.
Clustering of the different isolates
The genetic variation occurring in plant pathogenic
bacter ia due to mutations, intra-genomic re-
arrangements and horizontal gene transfer mechanisms
may result change in certain physiological activities of
the causal bacterium. Due to this variation, the isolates
of the same pathovar may exhibit considerable diversity
in their physiological or biochemical properties. The result
of the biochemical tests were subjected to cluster
analysis based on unweighted pair group method of
average using software programme PAST ver 2.1.5
(Hammer et al., 2001). Binomial data was incorporated
in the form of ‘1’ in case of positive and zero in case of
negative test. Two major group of isolates designated as
group I and group II were obtained. Group I was further
subdivided into two subgroups viz. group IA and IB.
Similarly, group II was subdivided into two subgroups
viz. group IIA and IIB. Group IA consisted of 15 isolates
of test bacterium. The isolates belonging to this group
could utilize all the tested carbohydrates while they were
unable to hydrolyze starch. Group IA isolates were also
tolerant to all the antibiotics tested. Group IB differentiated
from group IA as the isolates belonging to group IB could
not liquefy gelatin (Table 3). Group II contained 28 isolates
which showed resistance to all the antibiotics tested.
However these isolates were sensitive to streptomycin
at the test concentration. Group IIA isolates showed
variable carbohydrate and starch utilization pattern, while
group IIB isolates could not hydrolyze starch at all (Table
3).
Different isolates of Dickeya zeae may exhibit
variation in different physiological and biochemical
properties due to modification in certain enzymatic
activities. The phenotypic dendrogram of the 62 isolates
of D. zeae collected from different maize growing areas
378 Indian Phytopathology 68 (4) : 375-379 (2015)

Fig. 1. Percent frequency of positive reactions in biochemical tests showed by different isolates of Dickeya zeae

Table 3. Grouping of Dickeya zeae isolates of on the basis of a panel of 27 biochemical tests

Group Designation of Isolates No. of Remarks

isolates

IA M6, M10, M11, M29, M30, M36, M47, M48, M49,
M50, M51, M55, M56, M57, M58
IB M1, M3, M4, M5, M14, M15, M16, M17, M21, M22,
M28, M31, M32, M35, M59, M60, Chilli-1, Chilli-2,
Rice-1
II A M2,M8,M9
II B M7, M13, M18, M19, M20, M23, M24, M25, M26,
M27, M33, M34, M37, M38, M39, M40, M41, M42,
M43, M44, M45, M46, M52, M53, M54
15 Insensitive to all antibiotics, utilize all tested
carbohydrates, starch hydrolysis negative
19 Insensitive to all antibiotics, Carbohydrate utilization
variable, starch hydrolysis negative, Gelatin liquefaction
negative and methyl red positive
3 Streptomycin Sensitive , Carbohydrate utilization
variable, starch hydrolysis variable
25 Sensitive to streptomycin, utilize all tested
carbohydrates, starch hydrolysis negative

of Punjab was generated on the basis of their biochemical
assays and antibiotic sensitivity. The populations of soft
rot bacteria are regularly assessed for the biochemical
and physiological variations that affect the phenotype of
a particular strain/isolate. Kaneshiro et al. (2008) used a
panel of six phenotypic tests (KOH, YDC colony
morphology, O/F, pectolytic ability on CVP, indole
production, and indigoidine production) to characterize
33 isolates of E. chrysanthemi from pineapple. Janse
and Ruissen (1988) characterized 41 strains of E.
chrysanthemi from different hosts including maize using
physiological, biochemical, serological and pathogenicity
tests and showed differential utilization of carbohydrates
among the 41 strains tested. Similarly, Mohammed and
 Indian Phytopathology 68 (4) : 375-379 (2015)
Selman (2013) observed variation in carbohydrate
utilization pattern among the Erwinia strains infecting
potato crop. Similar findings were also reported from
other plant pathogenic bacteria (Adhikari and Mew, 1985;
Hunjan et al., 2012; Shenge et al., 2007). It can be
concluded from the current observations that Punjab
isolates of D. zeae vary in their physiological activities.
These studies will be further augmented by studying the
pathotypic variability in these isolates of D. zeae.
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Received for publication: July 27, 2015
Accepted for publication: September 11, 2015